2- or 3-dimensional geometric structures

ABSTRACT

Nucleic acid analogs provide a particularly useful tool for the preparation of complex polymeric structures of defined geometry because they are relatively stable to reaction conditions for the preparation of such structures and provide the opportunity to induce reactive groups which would not be possible with usual nucleic acids.

This is a divisional of application Ser. No. 09/087,514 filed May 28,1998 now U.S. Pat. No. 6,310,179.

Subject of the present invention is a method for constructing a 2- or3-dimensional defined polymeric geometric structure from oligomericelements, such geometric structures as well as the use of nucleic acidanalogues in assembling of supramolecular structures of defined form.

Living organisms are build from biomolecules particularly by definedself association of such biomolecules (lipids, protein complexes and DNAdouble helix). Such supramolecular structures occurring in livingorganisms are relatively unstable outside of these organisms, becausethe biomolecules have only limited affinity and are biologicallydegradable.

Lehn (Science 260, 1762-1763, 1993) describes the use of artificial selfassembled complexes from low molecular weight organic molecules andmetal ions.

It is further known that holes can be created in Longmuir-Blodget-layersduring cluster formation. Such artificial systems are based on theassociation of uniform molecules having no or small differences.

In nature complex structures are made up from macro molecules ofdifferent tertiary structures, based upon uniform basic structures likeamino acids and nucleotides. Nucleic acids of complementary sequenceform helical chains which can form later in-vivo special structures andnetworks.

In Angew. Chem. Int. Ed. Engl. 1997, 36, 7, 735-739 a supramolecularstructure based on macrocycles is disclosed. In J. Am. Chem. Soc. 1997,119, 852-853, control of stereochemistry in supramolecular architectureis described.

In J. Vac. Sci. Technol. A 12(4), 1895-1903, there is described theself-assembly of DNA molecules to form a 2-dimensional latice. Suchlatices are formed using DNA molecules the nucleotide sequence of whichis chosen such that four or more of such DNA molecules have a preferredassembly to form a junction. There is explicit explanation in thisdocument how the sequences of the DNA molecules involved has to bechosen. Further there is at least theoretical disclosure how a3-dimensional object, like a cube, can be created from 6 cyclic DNAmolecules. Again, the sequences of the DNA molecules are chosen suchthat the structure is stabilized and defined by the formation of doublestranded DNA along the edges of the cube.

In DE-A-3924454 and U.S. Pat. No. 5,561,071 there is described the useof self-assembled double stranded DNAs for forming conducting elements,for example elements used in electronic chips. The disclosure of thisreference is incorporated by reference into the present specification.

The supramolecular structures prepared by self-assembly of DNA have nowbeen found to be so unstable under in vitro conditions that, for exampleelectronic chips prepared by them are not reliable.

In WO 92/20702 there are disclosed purely synthetic oligomeric moleculeswhich are capable of binding to complementary nucleic acids with veryhigh affinity. This can be used for either therapy within the human bodyor in the diagnosis of nucleic acids in vivo or in vitro. Peptidenucleic acids (PNAs) as described in this reference are characterized byhaving a non-naturally occurring backbone having attached nucleobases atdefined positions, such that these nucleobases can hydrogen bond to thecomplementary bases on a DNA strand, thus forming a double or triplestranded complex.

It was therefore an object of the present invention to provide highmolecular weight supramolecular structures in a more reliable way.

It was an alternative or additional object of the present invention toprovide more stable high molecular weight supramolecular structures in apredictable way.

In a further object of the present invention to provide new materialsbased on supramolecular structures in an intended way, which can be usedin computer chips, in roboting, such as robot arms in nanometer scale,in new materials/polymers with conductivity and/or insulator properties.

Subject of the present invention is therefore a method for constructinga 2- or 3-dimensional defined polymeric geometric structures comprisingthe steps of combining a first oligomeric element having boundrecognition elements with a second oligomeric element having boundrecognition elements capable of recognizing the recognition elements ofsaid first oligomeric element at binding conditions, wherein saidrecognition elements are heterocyclic moieties recognizing otherrecognition elements via hydrogen bonding and wherein said recognitionelements of said first and second oligomeric element are bound to spaceddefined locations of a peptide bond containing backbone. A furthersubject of the present invention are such polymeric geometricstructures.

FIG. 1 shows the structure of a self-assembled 2-dimensional geometricstructure prepared by self-assembly of three different peptide nucleicacids.

A defined geometric structure according to the present invention is astructure having a defined expansion and which can be drawnschematically. Examples of such defined geometric structures arelatices, junctions, cubes and branched molecules as described in theabove mentioned prior art. Therefore, the invention can be used innano-engineering to create intended structures. While the prior artdescribes DNA for making up the structure, the present invention isdirected to the use of oligomeric elements comprising a peptide likebond containing backbone. The geometric structures preferably contain atleast one branching point. A branching point is defined to be thelocation where three or more arms meet. At least one of these armscomprises a segment wherein one strand of this arm is bound to a strandof a further oligomeric element. The oligo- or polymeric geometricstructure according to the present invention comprise at least twooligomeric elements. However, it is preferred that such a structurecontains 6 or more, preferably between 8 and 1 Million oligomericelements. In this definition oligomeric structures will contain from 2to 20 and polymeric structures more than 21 oligomeric elements. Theseoligomeric elements can be of the same kind, for example only differingin the sequence of the recognition elements, preferably, however, theoligomeric elements are of a different kind, for example differing insequence and in molecular structure, for example some of them beingmodified further or being based on different backbones or moietiesattached.

An oligomeric element according to the invention is defined to containaffinity moieties, such as alkyl, aryl, aromatic or/and heterocyclicmoieties recognizing other heterocyclic molecules via van-der-Waalsinteraction, π-stacking, water exclusion or hydrogen bonding. Saidaffinity moieties are bound to spaced defined locations of a polyamidebackbone. The backbone is generally a non-naturally occurrin backbone.The backbone preferably contains repetitive monomeric subunits, suchsubunits being covalently bound together, preferably using amide bondformation. While it is much preferred to use only one kind of monomericsubunit in the backbone, it is possible to use different subunits and/ordifferent bonds within the backbone either mixed individually or asstretches containing several identical subunits, as described in WO95/14706, EP 700928, EP 646595 and EP 672677.

Oligomeric elements having both a non-natural backbone part as well asan excessable oligonucleotide and can be used to postmodify thegeometric structure after assembly. Thus, it is possible to attachfurther mononucleotide units to the end of the oligomeric unit, forexample as described in EP 720615.

Preferred oligomeric elements, such as peptide nucleic acids (PNAs), aredescribed in WO 92/20702. Such compounds comprise a containing polyamidebackbone bearing a plurality of heterocyclic moieties that areindividually bound to amine atoms located within said backbone.

Preferred peptide nucleic acids are shown in formula I:

wherein

n is an integer of from at least 3,

x is an integer of from 2 to n−1,

each of L¹-L^(n) is a ligand independently selected from the groupconsisting of hydrogen, hydroxy, (C₁-C₄)alkanoyl, naturally occurringnucleobases, non-naturally occurring nucleobases, aromatic moieties, DNAintercalators, nucleobase-binding groups, heterocyclic moieties,reporter ligands and chelating moieties, at least one of L¹-L^(n)containing a primary or secondary amino group;

each of C¹-C^(n) is (CR⁶R⁷)y (preferably CR⁶R⁷, CHR⁶CHR⁷ or CR⁶R⁷CH₂)where R⁶ is hydrogen and R⁷ is selected from the group consisting of theside chains of naturally occurring alpha amino acids, or R⁶ and R⁷ areindependently selected from the group consisting of hydrogen,(C₁-C₆)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C₁-C₆)alkoxy,(C₁-C₆)alkylthio, NR³R⁴ and SR⁵, where R³ and R⁴ are as defined below,and R⁵ is hydrogen, (C₁-C₆)alkyl, hydroxy, (C₁-C₆)alkoxy, or(C₁-C₆)alkylthio-substituted (C₁-C₆)alkyl or R⁶ and R⁷ taken togethercomplete an alicyclic or heterocyclic system; or C¹-C″ is CO, CS, CNR³;

each of D¹-D^(n) is (CR⁶R⁷)_(z) (preferably CR⁶R⁷, CHR⁶CHR⁷ or CH₂CR⁶R⁷)where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z being atleast 2, preferably greater than 2, but not more than 10;

each of G¹-G^(n−1) is —NR³CO—, —NR³CS—, —NR³SO—or —NR³SO₂—, in eitherorientation, where R³ is as defined below;

each of A¹-A^(n) and B¹-B^(n) are selected such that:

(a) A¹-A^(n) is a group of formula (I/A), (I/B), (I/C) or (I/D), andB¹-B^(n) is N or R³N⁺; or

(b) A¹-A^(n) is a group of formula (I/D) and B¹-B^(n) is CH;

 wherein:

X is O, S, Se, NR³, CH₂ or C(CH₃)₂;

Y is a single bond, O, S or NR⁴;

each of p and q is zero or an integer from 1 to 5, (the sum p+q beingpreferably not more than 5);

each of r and s is zero or an integer from 1 to 5, (the sum r+s beingpreferably not more than 5);

each R¹ and R² is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl which may be hydroxy- or (C₁-C₄)alkoxy- or(C₁-C₄)alkylthio-substituted, hydroxy, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio,amino and halogen; and

each R³ and R⁴ is independently selected from the group consisting ofhydrogen, (C₁-C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted(C₁-C₄)alkyl, hydroxy, (C₁-C₆)-alkoxy, (C₁-C₆)-alkylthio and amino;

Q and I are independently selected from the group consisting of NH₂,CONH₂, COOH, hydrogen, (C₁-C₆)-alkyl, O—(C₁-C₆)-alkyl, amino protectedby a amino protecting groups, reporter ligands, intercalators,chelators, peptides, proteins, carbohydrates, lipids, steroids,nucleosides, nucleotides, nucleotide diphosphates, nucleotidetriphosphates, oligonucleotides, including both oligoribonucleotides andoligodeoxyribonucleotides, oligonucleosides and soluble and non-solublepolymers as well as nucleic acid binding moieties and

each of x1 and y1 is an integer of from 0 to 10.

Most preferred nucleic acid binding compounds comprise at least onemonomeric subunit of the general formula II:

wherein

L is a ligand as defined above for L¹-L^(n),

k, l and m is independently zero or an integer from 1 to 5,

p is zero or 1, and

R⁷ is selected from the group consisting of hydrogen and the side chainsof naturally occurring alpha amino acids.

Preferred recognition elements are nucleobases, like naturally occurringnucleobases, like A, G, C, T and U, rare bases, like inosine,5-methylcytosine or thiouracil, as well as any non-naturally occurringanalogues, like 7-deaza-dGTP, bromothymine and azaadenines. Theserecognition elements are able to recognize corresponding recognitionelements on another oligomeric element, as known in the art. The presentinvention thus provides the possibility to create intended structures,with high specificity, for example based on sequences of recognitionelements.

Hydrogen bonding is a way of binding, particularly as occurring betweentwo strands of a nucleic acid, including Watson-Crick base pairing andHoogsteen base pairing.

The recognition elements are bound to specified and constant locationson the backbone, preferably separated by between 4 and 8 interveningatoms. The preferred atom of attachment is a nitrogen atom.

The oligomeric elements used in the present invention can be prepared bymethods as described in the above documents. The use of peptide likebonds within the backbone provides a good opportunity of easy synthesisusing DNA or peptide synthesizers. When prepared, the oligomericelements can be coupled to other moieties that are intended to beincluded in the geometric structure of the present invention, such asrecognizing moieties, moieties that can be recognized, catalyticallyactive moieties, labels or chemically reactive or activatable moieties.Recognizing moieties and moieties that can be recognized are moietiesthat can recognize and preferably bind to an other component. Examplesare immunologically reactive compounds, antibodies, antigens, andpreferably peptide epitomes containing moieties, like polyhaptens orcyclic peptides. Such moieties can also connect oligomeric elements, forexample can one end of a cyclic peptide be linked to a first oligomericelement and the other end be linked to a second oligomeric element. Thiscan be accomplished by synthesizing such conjugate before introducinginto the polymeric structure, for example in a peptide synthesizer.

Catalytically active moieties are enzymes, preferably polymeric enzymes,for example aggregates of a large numbers of enzymes, bound togethercovalently. A variety of labels can be conjugated to the oligomericelement, for example fluorescence labels, dyes, or even metals or othersolid particles. Preferred are reactive groups.

Reactive groups useful in the present invention are groups that can bindcovalently to other groups, like recognizing moieties as defined above,preferably, groups that can be used to crosslink oligomeric elements orto bind any further moieties to the oligomeric elements before or afterassembly of the polymeric structure. Such crosslinking moieties are forexample arylazide, acylazide, diazirines, ketones, quinones, andpsoralens. The preparation of oligomeric elements useful in the presentinvention is disclosed in detail in WO 92/20702 and U.S. Pat. No.5,539,082. These references are incorporated herein.

As mentioned above the use of chimeric elements offers the possibilityto extend shorter oligomeric elements by, for example, enzymaticextension reactions using mononucleotides. Another example of extensionis chemical ligation of oligomeric subunits by reaction of a thioesteron one oligomer with a thiol group on another oligomer, also containingan amino group close to the thiol group. The thioester exchange willlink the two oligomers together. Then the amino group on the secondoligomer will interchange the newly formed thioester to form an amidebond (Canne et al. J. Am. Chem. Soc. 1996, 118, 5891-5896). Anotherexample of chemical ligation is the combination of an activated group ofone oligomer subunit with a functional group on the same or anotheroligomeric subunit. This reaction can be performed by an activatedcarboxylic acid derivative on one oligomer and an amino group on thesame or on an other oligomer.

Covalent joining of two segments can also be performed by usingduplexes/triplexes having overhanging ends. The overhanging end of oneduplex can then hybridize with the overhanging end of the otherduplex/triplex, thus forming a duplex with double length. By employing arecognition moiety capable of making a crosslink (see above) theoverhanging region can be covalently joined.

In a preferred mode of the method of the present invention at least twooligomeric elements are combined under conditions suitable for bindingthe oligomeric elements together by hydrogen bonding via the recognitionelements. Suitable conditions for peptide nucleic acid are disclosed inWO 92/20703. One of the biggest advantages of the use of PNA in thepresent invention is that they can bind via base pairing to nucleicacids or other PNA oligomers in non-physiological media, for example inwater without or with very low salt content.

PNA having bound chelating moieties are disclosed in WO 95/14708. ThesePNA oligomers further give the opportunity to bind metal ions (thusdoping the geometrical structure with metal ions) for increasingelectric conductivity within such oligo/polymeric structure. PNA havingattached peptide moieties are disclosed in WO 95/16202. These compoundsprovide the opportunity to functionalize the geometric structureaccording to the present invention by post modification, for exampleenzymatic modification, or to build up new or additional associationstructures, like the use of antibodies to bind to peptide antigens bymaking a “bridge” structure.

Contrary to the recognition between nucleic acids, the distances ofaffinity moieties in the sequence can be chosen more freely, because itis not necessary that, especially in the case wherein the first andsecond oligomeric element are made up of the same kind of backbone, torecognize nucleic acids, as in methods for nucleic acids determination.Therefore, the present invention provides a much more flexible way ofconstructing geometric structures than structures prepared by usingnaturally occurring DNA. However, it may be preferred, that thedistances between recognition elements within the segments of eacholigomeric element are at least compatible or similar, and preferablywithin segments of oligomeric elements designed for binding together viahydrogen bonding, and the distances between said recognition elementsare such that π-stacking and/or water exclusion of the recognitionelements is still possible. Favorable interstrand interaction can bereadily determined by determining the melting temperatures (T_(m)) ofthe double/triple stranded product produced by the first and secondoligomeric element. The higher the T_(m), the higher the interstrandaffinity.

A 1-dimensional structure is a structure having no branching points,i.e. a linear structure.

A 2-dimensional structure according to the present invention is forexample a structure having linear structures oriented in one layer,wherein said linear structures are preferentially connected to eachother at branching points. A 2-dimentional structure can be straight,curved or ring formed. Rings may be linked together as in a chain orcatenants.

A 3-dimensional structure can be defined as a 2-dimensional structuresextended into more than one layer or containing more than one branchingpoint. An example of 3-dimensional structure is a cube, a tube, orspiral shaped structure.

The geometric structures constructed according to the present inventionare of high interest in the preparation of nano-structures andnano-engineering. The development of nano-structures is of interestbecause of the small space required for advanced instruments and therequirements of small amounts of material compared to macroscopicstructures. The development into nano-structures can best be explainedin the field of computers. While the first computers required largespace because the underlying hardware had extended dimensions,development of semiconductor technology made it possible to createcomputers having very high capacity but requiring only limited space. Itis now recognized that the presently used photolithographic modes forpreparing computer chips limits the further downscaling of chips.Nano-structures as produced in the present invention now provide forthese smaller dimensions necessary for the construction of evendownscaled computer chips.

This possibility now enables the construction of wires or conductingelements as small, preferably in diameter, as double stranded nucleicacid analogues, the dimensions of which are in about the same range asdouble stranded nucleic acids. However, by including single strandedsegments into the geometric structure, it is possible to even downscaleany wires to the strength of single stranded nucleic acid analogues oreven simple organic molecules, for example carbohydrids.

The geometric structures according to the present invention can beprepared by combining two or more oligomeric elements (either extendedor not extended), under conditions wherein they can bind in a specificor non-specific way using the recognition elements. When specificjoining is intended, then natural or non-natural nucleobases can be usedwhich are capable of base pairing (a process generally calledhybridization). When non-specific joining is intended, then duplexformation takes place preferentially by interstrand π-stacking and/orwater exclusion.

In a first application of the geometric structure of the presentinvention the structure is therefore used as a mask to create a definedpattern, for example on a surface. Fixating of the nucleic acid analoguemay be by passive adsorption to the surface but can also be performed bybond formation (ionic or covalently) between the analogue and thesurface. Such bond can be formed by thermal or photochemical means.

The pattern created on the surface can be used to cover or coat part ofor all remaining parts (for example on said surface), not covered bysaid structure by a material, for example with doped silicon, forexample using metallo organic chemical vapour deposition, removing thegeometric structure containing the assembled oligomeric elements, andthen applying to the now unprotected part of the surface a secondmaterial, for example a conductor as doped gallium arsenide or dopedsilicon. Therefore the geometric structure of the present invention canbe designed as a network or a lattice, thus enabling the definedproduction of chips. A part of said conceptual considerations forpreparation of computer chips from networks or latices can betransferred in analogy from U.S. Pat. No. 5,561,071 which to this end isincorporated herein by reference.

The use of nucleic acid analogues and especially peptide nucleic acidsin this field has considerable advantages. Nucleic acid analogues aremore stable than nucleic acids under the conditions of coating surfaceswith metals. Especially in the field of the preparation of computerchips where one break within the geometric structure, for example bybreaking a glycosidic or phosphate bond in one of the nucleic acids,would have the severe consequence on the electric current that thencould not flow through the “wire”.

PNA are nucleic acid analogues that in preferred embodiments do notcontain glycosidic or/and phosphate bonds, and as such is much morestable, which reduces the risk of strand breaks considerably compared tonucleic acids. This makes the chips prepared using nucleic acidanalogues far more reliable in use. In addition nucleic acid analogues,as PNA, can be modified as described above in a more flexible way, dueto their relatively flexible way for introducing chemically reactivesites.

In a second embodiment, the supramolecular geometric structures preparedaccording to the present invention can be used as scaffolds for electrontransfer and thus lead electric current themselves. It was nowdetermined that double strands constructed using oligomeric elements ofthe present invention, particularly if an uninterrupted π-stack ofheterocyclic moieties or other aromatic/conjugated systems is containedwithin the double strand, can act as a nano-structural conductor forelectrons (see for example U.S. Pat. No. 5,591,578 or C & EN, Feb. 24,1997, Angew. Chem. Int. Ed. Engl. 1997, 36, 7, 735-739 or J. Am. Chem.Soc. 1997, 119, 852-853). In a simple application, it is therebypossible to connect two macroscopic electrodes via an electron transferscaffold, “wire”, composed of a double strand of oligomeric elements.Such electrodes can be metallic surfaces, to which one of the oligomericelements of the geometric structure is attached directly, for examplecovalently, or indirectly, for example by absorbing the geometricalstructure or an oligomeric element to the electrode surface.Immobilization of PNA to a carbon electrode is described in J. Am. Chem.Soc. 1996, 118, 7667-7670. The content of this publication isincorporated by reference as it is directed to the preparation of anucleic acid analogue attached to an electrode and the measurement ofpotentiograms of double stranded nucleic analogues bound to theelectrode.

Using the present invention, it is possible to connect two electrodes,each of them being prepared according to the prior art, by assembling ageometric structure according to the invention. This can be made in alinear way, as described above, or can include more partial surfaces,for example separated electrodes, which can be connected in a directedway using specific sets of oligomeric elements. For example it ispossible to provide a surface with an array of electrodes, eachelectrode having attached a specific oligomeric element or even anextended polymeric structure and thereafter connecting selectedelectrodes by connecting the oligomeric elements to create a geometricstructure according to the invention or arms of an extended geometricstructure of one electrode with an oligomeric element or an extendedgeometric structure of another selected electrode. In this way,customer-designed electrodes and structures can be prepared. Suchdevices containing an array of electrodes can be produced by selectivelyapplying one or more small droplets of a solution containing thesestructures or oligomeric elements to the surface of each electrode. Anyexcess of compounds may be washed away. Thereafter the connectionsbetween the selected electrodes can be established as described above.

It is further possible to postmodify any geometrical structure producedaccording to the method of the present invention by crosslinking or evenenzymatic extension. Crosslinking can be effected by irradiating ageometric structure having incorporated a photochemically active moiety,like acridine, arylazide, acylazide, diaziridines, ketones, quinones orpsoralenes with light of an appropriate wavelength for activating thecrosslinking reaction. Crosslinking can also be made by thermochemicalmeans, like in polymer preparation by radical or electrophilic chainreactions using double bond containing moieties, like acrylates orstyrenes, or by activation of functional groups like carboxylic acidswhich, when activated, are capable to react with other functional groupslike amines. In this way additional branching points or eveninterstrand/duplex stabilization of double stranded parts can beaccomplished. It is further possible to dope any negatively chargedmoieties in the geometrical structure by ions, for example silver ions,analogously to the method disclosed in Nature, vol. 391, p. 775, 1998,to which reference is made for the preparation and use of such dopedstructure.

Three dimensional structures can be made in a consecutive manner byadding 2-dimensional structures on top of each other, which forms anintegrated multilayer structure forms structure.

Modification of the conformation of a 3-dimensional structure can bedone by using special photochemical groups, like azabenzenes, stilbene,which isomerise (e.g. cis/trans isomers) upon irradiation.

The 2- or 3-dimensional structures can be preformed in solution beforeaddition to e.g. a solid phase or made directly on the surface by firstadding one strand of a duplex to the surface and then adding thecomplement strand so the duplex is formed on the solid surface. Theprecipitated structure may retain its structure as in solution, e.g.tertiary form as a helix, or it may change structure on the surface froma helix to a duplex with the form as a ladder.

Furthermore another advantage of using PNA is that the molecules allowfor certain sequences to form stable triplexes (PNA-DNA-PNA, orPNA-PNA-PNA). This binding motif allows to prepare much more integratedstructures than e.g. DNA.

Further subject of the present invention therefore is the use ofcompounds containing or being prepared by using a monomeric subunit ofthe general formula

wherein

X is an amino protecting group and Y is a carboxyl protecting group,

R₇ is a moiety containing a functional group with a positive or negativecharge, for example a carboxyl group, phosphate group or ammonium group,or is a moiety capable of complexing or chelating metal ions, or is areactive group, for example for inducing covalent crosslinking, forexample mercapto, maleinimido, quinone, nitrene or carbene or asdescribed above and L is an affinity moiety preferably heterocyclicmoiety or a reactive aromatic moiety, for example an aromatic azide orquinone for the preparation of supramolecular structures of defined formon the basis of base sequence specificity or base non-specificity,particularly for the construction of new materials (e.g. new polymers),capable of base pairing to nucleobases, conducting networks and computerchips, and for crosslinking such structures, or binding recognizingmoieties to such structures.

Nucleic acid analogues provide a particularly useful tool for thepreparation of complex polymeric structures of defined geometry becausethey are relatively stable to reaction conditions for the preparation ofsuch structures and provide the opportunity to introduce reactive groupswhich would not be possible with usual nucleic acids.

A further advantage of the use of nucleic acid analogues is thepossibility to provide them with better solubility in organic solventsand their relatively lower solubility in water compared to nucleicacids. Furthermore, the lipophilic nature of the nucleic acid analoguesprovides much higher affinities for many surfaces.

In the following a specific example for a method of the presentinvention is outlined.

EXAMPLE 1

Preparation of Nucleic Acid Analogues

Peptide nucleic acid is nucleic acid analogue having aN-(2-aminoethyl)-glycine backbone, with the bases linked to the centralN-atom by a 2-carboxylmethyl group. PNA is prepared by the methodsdisclosed in WO 92/20702 and U.S. Pat. No. 5,539,082. The sequences aregiven in each example.

EXAMPLE 2

Assembly of a Double Fork Geometric Structure Containing 6 Nucleic AcidAnalogues

According to example 1 the following peptide nucleic acid molecules areprepared:

1. H-TCA-CGT*-ACC-TAG-TCT*CT-TGC-AT*G-CAT-NH₂

2. H-CGA-TGC-T*AC-TCTCT*-CTA-GGT*-ACG-TGA-NH₂

3. (H-GTA-GCA-T*CG)²-(ATG-CAT*-GCA)¹-NH₂ (²Complementary to segment inbold in 2, and ¹ complementary to segment in bold in 1.

The following peptide nucleic acid molecules were prepared as controlmolecules

4. H-CTA-GGT*-ACG-TGA-NH₂ (Complementary to underlined segment in 1.)

5. H-TCA-CGT*-ACC-TAG-NH₂ (Complementary to underlined segment in 2.)

6. H-GTA-GCA-T*CG-NH₂ (Complementary to bold segment in 2.)

7. H-ATG-CAT*-GCA-NH₂ (Complementary to segment in bold in 1.)

T* indicates that the T-monomers at that position is composed of lysinecontaining backbone.

Molecules 1, 2, 3 are designed to form an oligomeric structurecontaining 6 PNA molecules as oligomeric elements (each of the three iscontained twice). The sequences are chosen such that there is specificand predetermined binding of the oligomeric elements 1, 2 and 3. Thegeometric structure formed has two branching points and four doublestranded segments.

Control Hybridizations

Measurement of the melting temperature (T_(m)) shows sequentialhybridization of the individual control parts indicating that allsegments are capable of hybridizing. Thus, hybridizing the 12-mers,control segments 4 and 5, to the complementary segments in either 1 or 2gives a clear transition at ca. 72° C. Hybridizing the 9-mer controlsegments (6 and 7) to either 1 or 2 gives a transition at ca. 60° C.Hybridization of the joining segment (3) to either 1 or 2 gives atransition at ca. 65° C. These experiments indicate that the stem has aT_(m) of about 73° C. (12 mer) and the fork part has a lower T_(m) ofca. 60° C. (9 mers).

Formation of Supramolecular Structures

Hybridization 1 and 2 to each other (single fork formation, joining twomolecules) gives a very clear transition at 73° C. with a large ΔOD justas the control segments showed. Mixing 1, 2 and 3 (forming the doublefork, joining 6 molecules) gives a clear transition at 73° C. and a weaktransition around 50-60° C. ΔOD in this experiment is the largestmeasured. This experiment indicates that the two hybridized stem parts(dimers, formed by duplex formation) of the structure can be joined bythe overlapping PNA (3). The overall structure is anticipated to be theintended “double fork”. In addition to the Tm measurements the increasedΔOD of the overall complex indicated that a larger structure is formed.

The supramolecular structures can be visualised by AFM (atomic forcemicroscopy)(ref. Hansma, H. G. & Hoh, J. Annu. Rev. Biophys. Biomol.Struct. 23, 115-139 (1994); Bustamante, C. & Rivetti, C. Annu. Rev.Biophys. Biomol. Struct. 25, 395-429 (1996); Han, W., Linsay, S. M.,Dlakic, M. & Harrington, E. R. Nature, 386, 563.). The structures cane.g. be prepared in solution and then applied to the readable surface,or prepared by consecutive adding the building blocks to the surface.Furthermore neutron diffraction can give indications of the average sizeof supramolecular structures in solution.

We claim:
 1. A method of constructing a predetermined two or threedimensional geometric structure comprising at least three oligomericelements, the method comprising binding the at least three oligomericelements to form a forked structure, each of the at least threeoligomeric elements independently comprising a peptide bond-containingbackbone and having a plurality of recognition elements bound to thebackbone at spaced and predetermined locations thereon, wherein each ofthe plurality of recognition elements independently comprises aheterocyclic moiety, and the at least three oligomeric elements arebound to each other via the recognition elements through hydrogenbonding, van-der-Waals interaction, π-stacking or a condensationreaction.
 2. The method of claim 1, wherein at least six oligomericelements are bound to each other in said binding step.
 3. The method ofclaim 1, wherein eight to one million oligomeric elements are bound toeach other in said binding step.
 4. The method of claim 1, wherein theat least three oligomeric elements each comprise a peptide nucleic acidcomprising a polyamide backbone bearing a plurality of heterocyclicmoieties which are bound at spaced and predetermined locations to amineatoms located within the backbone.
 5. The method of claim 1, wherein theat least three oligomeric elements each comprise a peptide nucleic acidof formula I

wherein n is at least 3; x is 2 to n−1; each of L¹-L^(n) isindependently selected from the group consisting of hydrogen, hydroxyl,C₁-C₄ alkanoyl, a naturally occurring nucleobase, a non-naturallyoccurring nucleobase, an aromatic moiety, a DNA intercalator, anucleobase binding group, a heterocyclic moiety, a reporter ligand and achelating moiety, wherein at least one of L¹-L^(n) contains a primary orsecondary amino group; each of C¹-C^(n) is independently selected fromthe group consisting of CO, CS, CNR³ wherein R³ is as defined below,(CR⁶R⁷)_(y), (CHR⁶CHR⁷)_(y) and (CR⁶R⁷CH₂)_(y), wherein R⁶ is hydrogenand R⁷ is selected from the group consisting of one of the side chainsof naturally occurring alpha amino acids, or R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen, C₁-C₆ alkyl, aryl,aralkyl, heteroaryl, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkylthio, NR³R⁴ andSR⁵, wherein R³and R⁴ are as defined below and wherein R⁵ is selectedfrom the group consisting of hydrogen, C₁-C₆ alkyl, hydroxy, C₁-C₆alkoxy and C₁-C₆ alkylthio-substituted C₁-C₆ alkyl, or R⁶ and R⁷, takentogether with the atoms to which they are bound, form an alicyclic orheterocyclic system; each of D¹-D^(n) is independently selected from thegroup consisting of (CR⁶R⁷)_(z), (CHR⁶CHR⁷)_(z) and (CH₂CR⁶R⁷)_(z),wherein R⁶ and R⁷ are as defined above; each of y and z is zero or aninteger from 1-10, wherein y+z is at least 2; each of G¹-G^(n−1) isindependently selected from the group consisting of —NR³CO—, —CONR³,—NR³CS—, —CSNR³—, —NR³SO—, —SONR³—,—NR³SO₂— and —SO₂NR³—, wherein R³ isas defined below; each of A¹-A^(n) and B¹-B^(n) are selected such that:(1) each of A¹-A^(n) is independently selected from the group consistingof a group of formula (I/A), (I/B), (I/C) and (I/D), and each ofB¹-B^(n) is independently N or R³N⁺, wherein R³ is as defined below, or(2) each of A¹-A^(n) is a group of formula (I/D) and each of B¹-B^(n) isCH;

 wherein X is selected from the group consisting of O, S, Se, CH₂ andC(CH₃)₂ and NR³, wherein R³ is as defined below; Y is selected from thegroup consisting of a single bond, O, S and NR⁴, wherein R⁴ is asdefined below; each of p and q is independently zero or an integer from1 to 5; each of r and s is independently zero or an integer from 1 to 5;each of R¹ and R² is independently selected from the group consisting ofhydrogen, hydroxy, C₁-C₄ alkoxy, C₁-C₄ alkylthio, amino, halogen andC₁-C₄ alkyl, which is unsubstituted or has a substituent selected fromthe group consisting of hydroxy, C₁-C₄ alkoxy and C₁-C₄ alkylthio; andeach of R³ and R⁴ is independently selected from the group consisting ofhydrogen, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkylthio, amino and C₁-C₄ alkyl,which is unsubstituted or has a substituent selected from the groupconsisting of hydroxy, C₁-C₄ alkoxy and C₁-C₄ alkylthio; Q and I areeach independently selected from the group consisting of NH₂, CONH₂,COOH, hydrogen, C₁-C₆ alkyl, O—C₁-C₆ alkyl, amino protected by an aminoprotecting group, a reporter ligand, an intercalator, a chelator, apeptide, a protein, a carbohydrate, a lipid, a steroid, a nucleoside, anucleotide, a nucleotide diphosphate, a nucleotide triphosphate, anoligonucleotide, an oligonucleoside, a soluble or non-soluble polymerand a nucleic acid binding group; and each of x1 and y1 is independentlyzero or an integer from 1 to
 10. 6. The method of claim 1, wherein atleast one of the at least three oligomeric elements comprises amonomeric subunit of formula II

wherein L is selected from the group consisting of hydrogen, hydroxyl,C₁-C₄ alkanoyl, a naturally occurring nucleobase, a non-naturallyoccurring nucleobase, an aromatic moiety, a DNA intercalator, anucleobase binding group, a heterocyclic moiety, a reporter ligand and achelating moiety and a ligand containing a primary or secondary aminogroup; each of k, l and m is independently zero or an integer from 1 to5; p is zero or 1; and R⁷ is selected from the group consisting ofhydrogen and one of the side chains of naturally occurring alpha aminoacids.
 7. The method of claim 1, wherein sets of recognition elementsare bound to each other, each set comprising a recognition element fromat least two different oligomieric elements, wherein at least onerecognition element from the set comprises a moiety independentlyselected from the group consisting of adenine, cytosine, guanine,thymine, uracil, inosine, 5-methylcytosine, thiouracil, 7-deaza-dGTP,bromothymine and azaadenine.
 8. The method of claim 1, wherein sets ofrecognition elements are bound to each other, each set comprising arecognition element from at least two different oligomeric elements,wherein at least one recognition element from the set comprises a moietyindependently selected from the group consisting of an immunologicallyreactive compound, an antibody an antigen and a peptideepitope-containing moiety.
 9. The method of claim 1, wherein each of theplurality of recognition elements on each of the at least threeoligomeric elements is separated from another of the plurality ofrecognition elements by between 4 and 8 intervening atoms on thebackbone of the oligomeric element to which the plurality of recognitionelements are bound.